Etiologic Risk Group: Risk group 4 (high individual risk but low community risk)
*/Disease Information (sort of like the Intro to your Mini Research Write up):
Pulmonary tuberculosis is caused by Mycobacteriumtuberuculosis. Tuberculosis is highly infectious with just droplets ejected from an infected person (cough, sneezes) able to grow M. tuberculosis in the lungs of another person. Though infectious, pulmonary tuberculosis is likely not to develop in a person with a strong immune system. However, due to poor healthcare in many developing and undeveloped countries, tuberculosis is the second most common infectious disease in the world. Macrophages phagocytose M. tuberculosis in the alveoli, where people with healthy immune systems successfully contain tuberculosis. However, in weak immune systems, white blood cells are not as efficient in stopping/slowing the growth of M. tuberculosis. Symptoms of tuberculosis include but is not limtied to coughing, chest pain, fatigue, and fever. Treatment for tuberculosis often takes between 6-9 months with mulitple drugs. There are many theories of why tuberculosis is difficult to treat; One possible reason is the observed asymmetrical division of M. tuberculosis, which leads to cells of varying growth rates, drug resistance, and development stage. It has been discovered that cells early in development are more susceptible to rifampin, and cell later in development is more susceptible to isoniazid and cycloserine. Traditional drug cocktails to treat tuberculosis may contain isoniazid, rifampin, ethambutol, and pyrazinamide (list is not exhaustive). New drugs are being developed due to developed drug resistance.
Druggable Target (list number or cite evidence from a paper/database showing druggable in another organism): 0.918
*EC#: 1.8.1.9
Link to BRENDA EC# page: http://www.brenda-enzymes.org/enzyme.php?ecno=1.8.1.9 -- Show screenshot of BRENDA enzyme mechanism schematic Enzyme Assay information (spectrophotometric, coupled assay ?, reagents):
Continuous Spectrophotometric Rate Determination
--- List cost and quantity of substrate reagents, supplier, and catalog #
0.1% (w/v) Thioredoxin Solution (Thioredoxin)
$190.00/mg, Sigma-Aldrich, T0910
100 mM 5'5-Dithio-bis(2-Nitrobenzoic Acid Solution) (DTNB)
$31.70/g, Sigma-Aldrich, D8130
Structure (PDB or Homology model)
-- PDB # or closest PDB entry if using homology model: 2A87
-- For Homology Model option:
n/a ---- Show pairwise alignment of your BLASTP search in NCBI against the PDB
n/a ---- Query Coverage:
n/a ---- Max % Identities:
n/a ---- % Positives
n/a ---- Chain used for homology:
n/a
Current Inhibitors: Protoporphyrin IX, (-)-epicatechin-gallate (ECG), (-)-epigallocatechin-3-gallate (EGCG); however, these were tested on rats and calves.
Expression Information (has it been expressed in bacterial cells): Yes.
Purification Method:
Various affinity chromatography techniques. His-tag-proteins by Ni–NTA column chromatography is a known successful technique for Trxb2.
Image of protein (PyMol with features delineated and shown separately):
*Amino Acid Sequence (paste as text only - not as screenshot or as 'code'):
MTAPPVHDRAHHPVRDVIVIGSGPAGYTAALYAARAQLAPLVFEGTSFGGALMTTTDVENYPGFRNGITGPELMDEMREQ
Thioredoxin reductase/trxB2
*NCBI Gene # or RefSeq#:
886232
*Protein ID (NP or XP #) or Wolbachia#:
NP_218430.1
*Organism (including strain):
Mycobacterium tuberculosis (strain ATCC 25618 / H37Rv)
Etiologic Risk Group:
Risk group 4 (high individual risk but low community risk)
*/Disease Information (sort of like the Intro to your Mini Research Write up):
Pulmonary tuberculosis is caused by Mycobacterium tuberuculosis. Tuberculosis is highly infectious with just droplets ejected from an infected person (cough, sneezes) able to grow M. tuberculosis in the lungs of another person. Though infectious, pulmonary tuberculosis is likely not to develop in a person with a strong immune system. However, due to poor healthcare in many developing and undeveloped countries, tuberculosis is the second most common infectious disease in the world. Macrophages phagocytose M. tuberculosis in the alveoli, where people with healthy immune systems successfully contain tuberculosis. However, in weak immune systems, white blood cells are not as efficient in stopping/slowing the growth of M. tuberculosis. Symptoms of tuberculosis include but is not limtied to coughing, chest pain, fatigue, and fever. Treatment for tuberculosis often takes between 6-9 months with mulitple drugs. There are many theories of why tuberculosis is difficult to treat; One possible reason is the observed asymmetrical division of M. tuberculosis, which leads to cells of varying growth rates, drug resistance, and development stage. It has been discovered that cells early in development are more susceptible to rifampin, and cell later in development is more susceptible to isoniazid and cycloserine. Traditional drug cocktails to treat tuberculosis may contain isoniazid, rifampin, ethambutol, and pyrazinamide (list is not exhaustive). New drugs are being developed due to developed drug resistance.
Link to TDR Targets page (if present):
n/a
Link to Gene Database page (NCBI, EuPath databases -e.g. TryTryp, PlasmoDB, etc - or PATRIC, etc.)
NCBI: http://www.ncbi.nlm.nih.gov/gene/886232#general-protein-info
Essentiality of this protein:
Txb2 is essential to M. tuberculosis due to being a major disulide reductase and functions in pyrimidine conversions.
Is it a monomer or multimer as biological unit? (make prediction athttp://www.ebi.ac.uk/msd-srv/prot_int/pistart.html):
Monomer
Complex of proteins?:
No
Druggable Target (list number or cite evidence from a paper/database showing druggable in another organism):
0.918
*EC#:
1.8.1.9
Link to BRENDA EC# page:
http://www.brenda-enzymes.org/enzyme.php?ecno=1.8.1.9
-- Show screenshot of BRENDA enzyme mechanism schematic
Enzyme Assay information (spectrophotometric, coupled assay ?, reagents):
Continuous Spectrophotometric Rate Determination
-- link to Sigma (or other company) page for assay (see Sigma links below)
http://www.sigmaaldrich.com/content/dam/sigma-aldrich/docs/Sigma/Enzyme_Assay/thioredoxinred.pdf
-- -or link (or citation) to paper that contains assay information
n/a
-- links to assay reagents (substrates) pages.
--- List cost and quantity of substrate reagents, supplier, and catalog #
Structure (PDB or Homology model)
-- PDB # or closest PDB entry if using homology model:
2A87
-- For Homology Model option:
n/a
---- Show pairwise alignment of your BLASTP search in NCBI against the PDB
n/a
---- Query Coverage:
n/a
---- Max % Identities:
n/a
---- % Positives
n/a
---- Chain used for homology:
n/a
Current Inhibitors:
Protoporphyrin IX, (-)-epicatechin-gallate (ECG), (-)-epigallocatechin-3-gallate (EGCG); however, these were tested on rats and calves.
Expression Information (has it been expressed in bacterial cells):
Yes.
Purification Method:
Various affinity chromatography techniques. His-tag-proteins by Ni–NTA column chromatography is a known successful technique for Trxb2.
Image of protein (PyMol with features delineated and shown separately):
*Amino Acid Sequence (paste as text only - not as screenshot or as 'code'):
MTAPPVHDRAHHPVRDVIVIGSGPAGYTAALYAARAQLAPLVFEGTSFGGALMTTTDVENYPGFRNGITGPELMDEMREQ
ALRFGADLRMEDVESVSLHGPLKSVVTADGQTHRARAVILAMGAAARYLQVPGEQELLGRGVSSCATCDGFFFRDQDIAV
IGGGDSAMEEATFLTRFARSVTLVHRRDEFRASKIMLDRARNNDKIRFLTNHTVVAVDGDTTVTGLRVRDTNTGAETTLP
VTGVFVAIGHEPRSGLVREAIDVDPDGYVLVQGRTTSTSLPGVFAAGDLVDRTYRQAVTAAGSGCAAAIDAERWLAEHAA
TGEADSTDALIGAQR
*length of your protein in Amino Acids
335 residues
Molecular Weight of your protein in kiloDaltons using the Expasy ProtParam website
Molar Extinction coefficient of your protein at 280 nm wavelength:
TMpred graph Image (http://www.ch.embnet.org/software/TMPRED_form.html). Input your amino acid sequence to it.
*CDS Gene Sequence (paste as text only):
*GC% Content for gene:
66.1%
*CDS Gene Sequence (codon optimized) - copy from output of Primer Design Protocol (paste as text only):
ATGACGGCGC CACCGGTCCA CGACCGTGCG CACCACCCCG TCCGGGATGT GATCGTGATC GGCTCGGGCC CCGCGGGATA CACCGCGGCC CTGTACGCCG CCCGCGCCCA GCTCGCCCCG CTGGTCTTCG AGGGCACCTC CTTCGGGGGT GCCTTGATGA CCACCACGGA TGTGGAAAAC TACCCGGGCT TTCGCAACGG AATTACCGGC CCGGAGTTGA TGGACGAGAT GCGCGAGCAA GCGCTGCGCT TTGGCGCCGA CCTGCGCATG GAGGATGTGG AATCGGTCTC GCTCCACGGG CCGCTGAAGT CGGTGGTGAC CGCGGACGGC CAGACGCACC GCGCCCGCGC CGTCATCCTG GCCATGGGCG CGGCCGCCCG GTACCTGCAG GTCCCGGGCG AGCAAGAGTT GCTCGGACGC GGCGTGTCGA GCTGCGCGAC CTGTGACGGC TTCTTCTTCC GGGACCAGGA TATCGCGGTG ATCGGCGGAG GTGACTCCGC CATGGAGGAA GCCACCTTCC TGACCCGGTT CGCGCGCTCG GTGACCCTGG TCCACCGGCG GGACGAGTTC CGGGCGTCGA AGATCATGCT CGATCGGGCC CGCAACAATG ACAAGATCCG GTTCCTGACC AATCACACGG TCGTGGCCGT CGACGGCGAT ACGACCGTCA CGGGCTTGCG CGTCCGTGAC ACCAATACCG GCGCCGAGAC CACCTTGCCG GTCACCGGTG TGTTCGTCGC GATCGGCCAC GAGCCGCGCT CGGGGCTGGT GCGCGAGGCG ATCGACGTGG ATCCGGACGG TTATGTCTTG GTGCAAGGTC GGACCACCTC CACGTCGCTG CCCGGCGTGT TCGCGGCCGG CGACCTCGTG GACCGTACCT ACCGCCAAGC GGTGACCGCC GCGGGGTCGG GCTGCGCCGC CGCGATCGAC GCCGAGCGCT GGCTCGCGGA ACACGCGGCG ACCGGAGAAG CCGACTCCAC CGACGCCTTG ATCGGAGCCC AGCGGTAG
*GC% Content for gene (codon optimized):
69.2%
Do Not Need this info for Spring (but still copy these lines to your Target page for now)
Primer design results for pNIC-Bsa4 cloning (list seqeunces of all of your ~40 nt long primers):
(link to DNA Works output text file - that should be saved in your Google Docs folder after you did the primer design protocol)
-- Ask a mentor, Dr. B, or a fellow researcher -how to link a GDocs file if you are not sure how to.
Primer design results for 'tail' primers (this is just 2 sequences):
**